JC virus (JCV) causes a fatal disease in the central nervous system (CNS), known as progressive multifocal leukoencephalopathy (PML) in patients with underlying immunosuppressive conditions, including leukemia, lymphoma and AIDS. PML in the era of the AIDS epidemic dramatically increased and is an AIDS associated illness. This disease is also steadily increasing among patients with autoimmune disorders, such as multiple sclerosis and Crohn's disease, who are treated with antibody-based drugs (natalizumab), which makes JCV as a risk factor for autoimmune disease populations. Our lab has considerable experience in studying the unique biology of JCV for a number of years. In recent years, however, we have focused our attention to the investigation of the functional roles of one of its regulatory proteins, Agnoprotein (Agno) (71 aa long), which is shown to play critical roles in the viral life cycle. In the absence of its expression, JCV is unable to sustain its productive cycle. We have made significant advances in understanding the biological functions of Agno in recent years. For instance, (i) Agno was found to form highly stable dimeric/oligomeric (multimeric) structures in vitro and in vivo and this is mediated by amino acids spanning from 28 to 39 which forms an amphipathic alpha-helix confirmed by our recent NMR studies. (ii) Agno undergoes a rapid degradation process and the viral replication levels significantly decrease if the alpha- helix region is altered by mutagenesis. (iii) Interestingly, the dimerization domain mutants were found to have profound defects in the alternative splicing process of the viral transcripts, suggesting new roles for Agno in regulation of viral RNA splicing. The initial splicing studies wit the dimerization domain mutants even led to the discovery of a novel and previously unpredicted open reading frame (new ORF) for JCV late transcripts, the significance of which needs to be uncovered for the JCV replication cycle. (iv) Moreover, Agno was also found to specifically bind to the JCV RNA and harbors a predicted nuclear export signal (NES). Furthermore, it specifically interacts with CRM1 and accumulates in the nucleus of the infected cells if they are treated with a CRM1 specific inhibitor, Leptomycin B. These findings also suggest novel regulatory roles for Agno in nucleo- cytoplasmic export of the viral RNA in a CRM1-dependent manner. Collectively, these findings provide us with a strong rationale to further investigate the novel regulatory roles of Agno in splicing and the nucleo- cytoplasmic export of JCV transcripts; and allowed us formulate our central hypothesis: Agno and its multimeric forms play important regulatory roles in the alternative splicing of the viral transcrips and their CRM1-dependent nucleo-cytoplasmic export. We propose to examine our hypothesis by thoroughly investigating the molecular mechanism(s) of the regulation of the both events by Agno. 1